ABSTRACT Aging is a main risk factor for intervertebral disc degeneration (IDD), the main cause of chronic low back pain. Age-related changes in intervertebral disc (IVD) cells, particularly in the nucleus pulposus (NP), are thought to compromise homeostasis and lead to tissue degeneration by mechanisms that are not well understood. We propose to study the role of the Forkhead box O (FOXO) family of transcription factors in IVD homeostasis during aging. The scientific premise of this proposal is that: i) aging is a main cause of IDD but the molecular mechanisms underlying IVD aging remain incompletely characterized, ii) FOXO are important regulators of lifespan and cell homeostasis but their role in IVD homeostasis is unknown, iii) the proposed studies will elucidate the function of FOXO in NP and have potential to generate fundamental new insight into IVD biology, aging and degeneration. This premise is supported by our preliminary studies that revealed a reduction in FOXO expression in degenerated human IVD and in mouse IVD during aging, and that conditional FOXO deletion in mice led to spontaneous IVD degeneration. In the present application we propose to test our hypothesis that FOXO1 and FOXO3 are critical factors in maintaining cellular homeostasis and viability of NP cells during aging and that their aging-related reduction is a driver of IVD aging and increases the risk for IDD development. The proposed studies will define the role of FOXO1 and FOXO3 in promoting NP homeostasis at the tissue, cellular and transcriptomic levels. In Aim 1, we will investigate the molecular mechanisms that regulate FOXO1 and FOXO3 expression as well as define the FOXO signaling network in in human NP cells to identify the precise pathways whereby FOXO promote NP cell function and survival. In Aim 2, we will investigate the age- related molecular and structural changes in the spines of mice with inducible, NP-specific deletion of FOXO1, FOXO3 or all FOXO isoforms. In Aim 3, we will test whether restoring FOXO function in mature NP ameliorates the severity of age-related degenerative changes in a transgenic mouse model with inducible, tissue-specific FOXO1 or FOXO3 overexpression. Successful completion of the proposed project will establish FOXO1 and FOXO3 as critical regulators of IVD homeostasis during aging. Outcomes from these studies will lead to the development of new pharmacological interventions aimed at preserving IVD structure and function during aging.